Mammalian cells such as CHO (Chinese Hamster Ovarian cells), NS0 and PerC6 cells are routinely employed within the biopharmaceutical industry to manufacture biopharmaceuticals. These cells are genetically engineered and then selected in such a way so as to ensure that high titre expression of the desired protein is observed when the resulting cell lines are cultured in bioreactors.
Currently all approaches to select the best secreting cell clones involve the screening of hundreds to thousands of transfected cells to identify preferred clones with optimal growth and production profiles (e.g. see Wurm, 2004, Nature Biotechnology 22, 1393-1398). All such methodology requires considerable time and effort. As a consequence there is ongoing need for improved methodology to select for high yielding and stable cell lines. Such methodology should be faster, less labour and less resource intensive than current methodology. Typical current cell line development protocols involve the delivery of desired expression and selection cassettes into a desired host cell followed by plating, selection and growth of resulting genetically engineered cells in serum and animal derived component free (ADCF) media. Such ADCF media, first developed in the early 1990s, are now regularly employed to select and grow recombinant cells producing biopharmaceutical proteins intended for clinical use. The use of such cell culture media for this purpose ensures the mammalian cell lines generated are not exposed inadvertently to adventitious or transmissible agents of known or unknown origin. The use of such ADCF media thus forms part of a strategy to ensure resulting cell lines and thereby biopharmaceutical cell banks, processes, manufacturing plants and final products are not contaminated by such adventitious and transmissible agents be they cellular, viral or protein in origin. The use of ADCF media as part of a strategy to ensure contamination-free manufacturing not only addresses a theoretical risk to patients and manufacturing plants: biological drug products have previously been adulterated with adventitious agents (e.g. Huang W T et al Pharmacoepidemiol Drug Safety 2010, 19(3):306-310) and manufacturing sites have previously been closed due to contamination with such agents (e.g. see Garnick R L Dev Biol Standard 1998: 93 pp 21-9 & FDA web site notification (June 2009) regarding Vesivirus contamination of a manufacturing plant's bioreactors). Further details on such considerations and how best to reduce risks are well documented in ICH Quality Guide Q5A “Safety Evaluation of Biotechnology Products Derived From Cell Lines of Human or Animal Origin” (Step 4 Version September 1999).
Certain cell screening methodologies designed to identify optimal genetically engineered producer cells employ flow cytometry in order to reduce time and effort. When using flow cytometry to select engineered cell lines that secrete high levels of desired recombinant protein, two approaches have been adopted.
The first approach involves the selection of cells in which a fluorescent co-marker (e.g. GFP) or an enzyme such as DHFR (when combined with a fluorescent substrate) is over expressed. For such selection methods to succeed, one must attempt to link the expression of the markers with expression of the desired protein products. This linkage is required to ensure that cells expressing high levels of the marker also express and secrete high levels of the desired protein product. Examples of such an approach include Yoshikawa T et al 2001 (Biotech and Bioeng 74: 5 pp 435-442), Meng Y. G et al 2000 (Gene 25:242 (1-2) 201-207), deMaria et al 2007 (Biotechnol Prog 23, 475-72) and US 2004/0148647. However, there are drawbacks to this approach, in particular the fact that one is primarily selecting for high levels of marker expression and this does not always correlate with high expression and secretion of desired protein product. This approach also results primarily in the selection of final cells expressing extremely large amounts of marker. This can be disadvantageous, since high expression of a marker will compete with the desired protein product for host cell transcription, translation and processing apparatus, and thus the ultimate yield of the desired protein may be reduced. Furthermore such marker proteins can also be toxic (Liu H S et al 1999 (Biochem Biophys Res Commun 260 (3): 712-7)) and thereby inhibit cell line growth and production.
The second approach involves the direct selection of cells that secrete high levels of desired protein product. For an overview of FACS in general, see Shapiro: Practical Flow Cytometry, Fourth Edition, 2003, Wiley-Liss, ISBN#9780471411253. In a broad sense there are two FACS based methods for selection of high expression clones directly.
The first ‘direct selection’ FACS-based method exploits the observed correlation between membrane bound levels of desired protein product with secretion levels. An example of this approach can be found in Marder P et al 1990 Cytometry 11: 498-505. In this study the authors stained the membrane of hydridoma cells with fluorescently conjugated anti-product antibodies and then sorted and selected the most highly fluorescent cells. They then demonstrated that the resulting sub-clones exhibited enhanced IgG secretion levels in comparison to the cells prior to sorting. Subsequent reports with different cell lines have demonstrated similar results (e.g. see Brezinsky S et al 2003. Jn of Immunol (2003) 141-155).
A related but more complex alternative to staining and then sorting on membrane levels of product involves the use of product entrapment approaches such as the gel microdrop (GMD) technique or matrix-based secretion assays. In such approaches, secreted antibody is retained by either cross linkage to the cell membrane or within gel microdrops, or immobilised on an artificial matrix on the cell surface prior to FACS sorting with anti-product antibody on levels of fluorescence.
For a recent review on cytometry based cell sorting methods discussing both indirect selection approaches (i.e. via use of a marker) and direct selection approaches (i.e., via labelling of desired product associated with the cells) see Carroll and Al-Rubeai 2004 (Expert Opin Biol Ther 4: 1821-9) and Browne and Al-Rubeai 2007 (Trends Biotech, 25(9), 425-32).
However, whilst both direct and indirect labelling methods as described above are now proven successful, none to date are ideal for use in selecting high producing cell lines suitable for the manufacture of biopharmaceuticals. This is because, if the indirect approach is employed, one selects primarily for cells producing high marker levels as discussed above. Alternatively, all the direct selection methods to date employ animal derived anti-product antibodies and supporting reagents (e.g. foetal calf serum (FCS) or bovine serum albumin (BSA)), which are undesirable in the development and manufacture of biopharmaceuticals. Such animal reagents are also regularly employed to support cell viability during harsh flow sorting and plating procedures. This will again increase the risk of exposure to adventitious agents for any cell line thereby generated. Additionally, animal derived reagents such as serum or BSA are typically used to block non-specific binding prior to incubation of cells with the desired anti-product binding reagent such as anti-serum.
Recently, Applebaum et al. (US 2010/0028904), described a method to screen and select for high expressing cells by plating the cells in methylcellulose (semi-solid media) containing fluorescent Protein A/G to detect product expression. However, considerable challenges still remain with such methodology. Significantly, the fluorescent Protein A/G does not necessarily bind the protein of interest on the cell surface but rather the secreted protein around the cell (resulting in a “halo” around the cell mass). Indeed, Protein A primarily binds to antibody and Fc fusion proteins in the constant domain and it remains unknown if the Protein A binding epitopes of any such targets are presented and available for binding on the cell membrane prior to product dissociation and full secretion from the cell. Thus, this method largely relies on labelling of the Fc-protein free of the cell, suspended in the semi-solid medium. The result is that the method cannot guarantee clonality of the lines picked. In addition, the conditions for the growth of cells in semi-solid media and the amount of fluorescent Protein A/G to be used may need to be established each time the method is used. The selection of cells from semi solid medium is also time consuming, as a period of time (normally at least seven days) is required to enable the cell to secrete sufficient quantities of product into the medium surrounding the cells to enable detection.
Moreover, the levels of target binding and thereby levels of fluorescence achievable are considerably reduced due to the lower avidity of Protein A, relative to higher avidity antiserum (such as a non-human anti-product antiserum). For these reasons Protein A is typically only employed as a secondary labelling reagent in conjunction with higher avidity primary reagents.
There is a desire in the art for improved cell selection methods.